Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials

Abstract

The present invention relates to processes for inserting DNA into eucaryotic cells, particularly DNA which includes a gene or genes coding for desired proteinaceous materials for which no selective criteria exist. The insertion of such DNA molecules is accomplished by cotransforming eucaryotic cells with such DNA together with a second DNA which corresponds to a gene coding for a selectable marker. This invention also concerns processes for producing proteinaceous materials such as insulin, interferon protein, growth hormone and the like which involve cotransforming eucaryotic cells with DNA which codes for these proteinaceous materials, growing the contransformed cells for production of the proteinaceous material and recovering the proteinaceous material so produced. The invention further relates to processes for inserting into eucaryotic cells a multiplicity of DNA molecules which includes genes coding for desired proteinaceous materials. The insertion of multiple copies of desired genes is accomplished by cotransformation with the desired genes and with amplifiable genes for a dominant selectable marker in the presence of successively higher amounts of an inhibitor. Alternatively, the insertion of multiple copies of desired genes is accomplished by transformation using DNA molecules formed by ligating a DNA molecule including the desired gene to a DNA molecule which includes an amplifiable gene coding for a dominant selectable phenotype such as a gene associated with resistance to a drug in the presence of successively higher amounts of an agent such as a drug against which the gene confers resistance so that only those eucaryotic cells into which multiple copies of the amplifiable gene have been inserted survive. Eucaryotic cells into which multiple copies of the amplifiable gene have been inserted additionally include multiple copies of the desired gene and may be used to produce multiple copies of proteinaceous molecules. In this way otherwise rare proteinaceous materials may be obtained in higher concentrations than are obtainable using conventional techniques.

What is claimed is: 
     
       1. A process for inserting foreign DNA I into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with said foreign DNA I and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA II being attached to bacterial plasmid or phage DNA. 
     
     
       2. A process in accordance with claim 1, wherein the DNA I or KNA II is attached to phage DNA and encapsidated in a phage particle. 
     
     
       3. A process in accordance with claim 1, wherein the DNA I or DNA II is attached to bacterial plasmid DNA. 
     
     
       4. A process in accordance with claim 3, wherein the bacterial plasmid DNA is derived from pBR322. 
     
     
       5. A process in accordance with claim 1, wherein said foreign DNA I codes for proteinaceous material which is not associated with a selectable phenotype. 
     
     
       6. A process in accordance wirh claim 5, wherein the proteinaceous material which is not associated with a selectable phenotype comprises interferon protein, insulin, a growth hormone, a clotting factor, a viral antigen or an antibody. 
     
     
       7. A process in accordance with claim 1, wherein said foreign DNA I is present in an amount relative to said DNA II which codes for a selectable phenotype in the range from about 1:1 to about 1000,000:1. 
     
     
       8. A process in accordance with claim 1, wherein said DNA II which codes for a selectable phenotype comprises the gene for thymidine kinase from herpes simplex virus. 
     
     
       9. A process in accordance with claim 1, wherein said DNA II which codes for a selectable phenotype comprises the gene for adenine phosphoribousyltransferase. 
     
     
       10. A process in accordance with claim 1, wherein said DNA II which codes for a selectable phenotype comprises a gene associated with drug resistance. 
     
     
       11. A process in accordance with claim 10, wherein the gene associated with drug resistance is the gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate. 
     
     
       12. A eucaryotic cell into which foreign DNA I has been inserted in accordance with the process of claim 1. 
     
     
       13. A mammalian cell into which foreign DNA I has been inserted in accordance with the process of claim 1. 
     
     
       14. A process for producing a proteinaceous material which comprises cotransforming a suitable eucaryotic cell in accordance with the process of claim 1, maintaining the cotransformed eucaryotic cell under suitable conditions permitting production of the proteinaceous material and recovering the proteinaceous material so produced. 
     
     
       15. A process in accordance with claim 14, wherein the suitable eucaryotic cell is a mammalian cell. 
     
     
       16. A process for producing a proteinaceous material which comprises cotransforming a suitable eucaryotic cell with foreign DNA I which codes for foreign proteinaceous material which is not associated with a selectable phenotype and with unlinked foreign DNA II which codes for a selectable phenotype not expressed by the eucaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucaryotic cells which have acquired the selectable phenotype, maintaining or culturing the cotransformed cells under suitable conditions permitting production of the proteinaceous material and recovering the proteinaceous material so produced. 
     
     
       17. A process in accordance with claim 16, wherein the eucaryotic cell is a mammaliam cell. 
     
     
       18. A process in accordance with claim 16, wherein the foreign DNA I is present in an amount relative to DNA II in the range from about 1:1 to about 100,000:1. 
     
     
       19. A process in accordance with claim 16, wherein the DNA II which codes for a selectable phenotype comprises the gene for thymidine kinase from herpes simplex virus. 
     
     
       20. A process in accordance with claim 16, wherein the DNA II which codes for a selectable phenotype comprises the gene for adenine phosphoribosyltransferase. 
     
     
       21. A process in accordance with claim 16, wherein the DNA II which codes for a selectable phenotype comprises a gene associated with drug resistance. 
     
     
       22. A process in accordance with claim 21, wherein the gene associated with drug resistance is the gene coding for a mutant dihydrofolate reductase which renders cells resistant to methotrexate. 
     
     
       23. A process in accordance with claim 16, wherein the eucaryotic cell is cotransformed with a multiplicity of foreign DNA I molecules.